p53 突变型在人类前列腺癌细胞中决定了对苯乙基异硫氰酸酯诱导的生长抑制的敏感性。
p53 mutant-type in human prostate cancer cells determines the sensitivity to phenethyl isothiocyanate induced growth inhibition.
机构信息
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington DC, 20007, USA.
Department of Biochemistry and Molecular and Cellular Biology, Georgetown University, Washington DC, 20007, USA.
出版信息
J Exp Clin Cancer Res. 2019 Jul 15;38(1):307. doi: 10.1186/s13046-019-1267-z.
BACKGROUND
We reported previously that phenethyl isothiocyanate (PEITC), a dietary compound, can reactivate p53 mutant in vitro and in SK-BR-3 (p53) breast xenograft model resulting in tumor inhibition. Because of the diversity of human cancers with p53 mutations, these findings raise important questions whether this mechanism operates in different cancer types with same or different p53 mutations. In this study, we investigated whether PEITC recuses mutant p53 in prostate cancer cells harboring different types of p53 mutants, structural and contact, in vitro and in vivo.
METHODS
Cell proliferation, cell apoptosis and cell cycle arrest assays were performed to examine the effects of PEITC on prostate cancer cell lines with p53 mutation(s), wild-type p53, p53 null or normal prostate cells in vitro. Western blot analysis was used to monitor the expression levels of p53 protein, activation of ATM and upregulation of canonical p53 targets. Immunoprecipitation, subcellular protein fraction and qRT-PCR was performed to determine change in conformation and restoration of transactivation functions/ inhibition of gain-of-function (GOF) activities to p53 mutant(s). Mice xenograft models were established to evaluate the antitumor efficacy of PEITC and PEITC-induced reactivation of p53 mutant(s) in vivo. Immunohistochemistry of xenograft tumor tissues was performed to determine effects of PEITC on expression of Ki67 and mutant p53 in vivo.
RESULTS
We demonstrated that PEITC inhibits the growth of prostate cancer cells with different "hotspot" p53 mutations (structural and contact), however, preferentially towards structural mutants. PEITC inhibits proliferation and induces apoptosis by rescuing mutant p53 in p53 contact (VCaP) and p53 structural (LAPC-4) mutant cells with differential potency. We further showed that PEITC inhibits the growth of DU145 cells that co-express p53 (structural) and p53 (contact) mutants by targeting p53 mutant selectively, but not p53. The mutant p53 restored by PEITC induces apoptosis in DU145 cells by activating canonical p53 targets, delaying cells in G1 phase and phosphorylating ATM. Importantly, PEITC reactivated p53 and p53 mutants in LAPC-4 and DU145 prostate xenograft models, respectively, resulting in significant tumor inhibition.
CONCLUSION
Our studies provide the first evidence that PEITC's anti-cancer activity is cancer cell type-independent, but p53 mutant-type dependent.
背景
我们之前报道过，苯乙基异硫氰酸酯（PEITC）是一种饮食化合物，可在体外和 SK-BR-3（p53）乳腺异种移植模型中重新激活 p53 突变体，从而抑制肿瘤。由于 p53 突变的人类癌症种类繁多，这些发现提出了一个重要的问题，即这种机制是否在具有相同或不同 p53 突变的不同癌症类型中起作用。在这项研究中，我们研究了 PEITC 是否可以在体外和体内重新激活具有不同类型 p53 突变（结构和接触）的前列腺癌细胞中的突变型 p53。
方法
细胞增殖、细胞凋亡和细胞周期阻滞实验用于检测 PEITC 对具有 p53 突变、野生型 p53、p53 缺失或正常前列腺细胞的前列腺癌细胞系的影响。Western blot 分析用于监测 p53 蛋白的表达水平、ATM 的激活以及经典 p53 靶标的上调。免疫沉淀、亚细胞蛋白部分和 qRT-PCR 用于确定 p53 突变体构象的变化和反式激活功能/抑制获得性功能（GOF）活性的恢复。建立小鼠异种移植模型以评估 PEITC 的抗肿瘤功效和体内诱导 p53 突变体重新激活的作用。对异种移植肿瘤组织进行免疫组织化学染色，以确定 PEITC 对体内 Ki67 和突变型 p53 表达的影响。
结果
我们证明，PEITC 抑制具有不同“热点”p53 突变（结构和接触）的前列腺癌细胞的生长，但对结构突变体的抑制作用更强。PEITC 通过以不同的效力挽救 p53 接触（VCaP）和 p53 结构（LAPC-4）突变细胞中的突变 p53，抑制增殖并诱导凋亡。我们进一步表明，PEITC 通过选择性靶向突变型 p53 而不是 p53 抑制共表达 p53（结构）和 p53（接触）突变体的 DU145 细胞的生长。PEITC 恢复的突变型 p53 通过激活经典的 p53 靶标、将细胞阻滞在 G1 期并使 ATM 磷酸化，在 DU145 细胞中诱导细胞凋亡。重要的是，PEITC 在 LAPC-4 和 DU145 前列腺异种移植模型中重新激活了 p53 和 p53 突变体，导致肿瘤显著抑制。
结论
我们的研究首次提供了证据，证明 PEITC 的抗癌活性是与癌细胞类型无关，但与 p53 突变体类型有关。
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